Borrelia burgdorferi (Bb), the Lyme disease spirochete, undergoes dramatic adaptive changes as it cycles in nature between its diverse tick and mammalian hosts. However, little is known regarding the genetic regulatory networks that modulate Bb's infectivity and virulence. Recently, we discovered a novel regulatory pathway in Bb, wherein one alternative sigma factor (sigma N, sigmaN, sigma54, RpoN) regulates the expression of another alternative sigma factor (sigma S, sigmaS, sigma38, RpoS) which, in turn, governs the expression of key membrane lipoproteins associated with borrelial virulence. This study proposes to build on this initial observation and investigate, at the genetic and molecular levels, the novel RpoN-RpoS regulatory pathway as it pertains to Bb's infectivity and virulence. In Specific Aim 1, we will identify the activator of the RpoN-RpoS regulatory pathway; studies will focus on examining a role for the "response regulator protein 2" (Rrp2) of Bb that is predicted to be a transcriptional enhancer-binding protein. In Specific Aim 2, we will examine the mechanism by which RpoN controls the expression of RpoS, with emphasis on (i) mapping the rpoS promoter and (ii) assessing the binding of RpoN and its activator (Rrp2) to the rpoS promoter and upstream regions. Studies in Specific Aim 3 will investigate the mechanism by which the RpoN-RpoS pathway regulates the expression of a prototypic differentially regulated lipoprotein, OspC. Emphasis will be placed on analyzing key features of the ospC promoter that engender its control by RpoN via RpoS. In Specific Aim 4, we will examine the influence of the RpoN-RpoS pathway on Bb's ability to infect animals, colonize ticks, be vector-transmitted, and cause disease. Finally, in Specific Aim 5, we will use DNA microarrays to identify more globally those genes of Bb influenced by the RpoN-RpoS regulatory pathway. These combined studies will be instrumental in further characterizing the novel RpoN-RpoS regulatory pathway in Bb and for potentially identifying infection- and/or virulence-associated genes that contribute to many aspects of Bb's complex parasitic strategy.